Address correspondence and reprint requests to Dr Jin-Chung Chen, Room 664, Department of Physiology and Pharmacology, Chang-Gung University, 259 Wen-Hwa 1st Road, Tao-Yuan, Kwei-Shan, Taiwan, R.O.C. 333. E-mail: firstname.lastname@example.org
The function of the D3 dopamine (DA) receptor remains ambiguous largely because of the lack of selective D3 receptor ligands. To investigate the function and intracellular signaling of D3 receptors, we established a PC-12/hD3 clone, which expresses the human D3 DA receptor in a DA producing cell line. In this model, we find that the D3 receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre-treatment with 3,6a,11, 14-tetrahydro-9-methoxy-2 methyl-(12H)-isoquino[1,2-b] pyrrolo[3,2-f][1,3] benzoxanzine-1-carboxylic acid, a D3 receptor preferring agonist, dose-dependently suppressed K+-evoked [3H]DA release in PC-12/hD3 cells but not in the control cell line. This effect was prevented by D3 receptor preferring antagonists GR103691 and SB277011-A. Furthermore, activation of D3 receptors significantly inhibits forskolin-induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin-dependent kinase 5 (Cdk5), dopamine and cAMP-regulated phosphoprotein of Mr 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol-3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D3 receptor inhibition of DA release. In a complimentary experiment, over-expression of Cdk5 potentiated D3 receptor suppression of DA release. Pertussis toxin, 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one and cyclosporine A also attenuated D3 receptor-mediated inhibition of DA release indicating that this phenomenon acts through Gi/oα and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D3 DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D3 DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.
The D3 dopamine (DA) receptor is exclusively expressed in the limbic forebrain, and postulated to be critically involved in mood, emotion, and diseases such as schizophrenia and bipolar disorder (Sokoloff et al. 2006). It has been suggested that in addition to serving as classical post-synaptic receptors, D3 receptors also function as pre-synaptic autoreceptors for controlling transmitter release and synthesis (Diaz et al. 2000). D3 receptor knockout mice exhibit hyperactivity and a higher basal DA level in the synapse suggesting that D3 receptors regulate DA release (Koeltzow et al. 1998; Joseph et al. 2002). However, reports of signaling downstream from D3 receptor activation are highly varied and the mechanism by which D3 receptors act to modulate synaptic DA levels is as yet largely unknown.
Pre-synaptic secretory machinery is centrally regulated by a series of calcium-dependent and protein phosphorylation events. Several protein kinases, such as protein kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II, were shown to participate in this secretory cascade (Sokoloff et al. 2006). Recently, cyclin-dependent kinase 5 (Cdk5) was revealed as a general enhancer of exocytosis in neuroendocrine cells (Fletcher et al. 1999) and pancreatic cells (Liu et al. 2001). Cdk5 is known to phosphorylate cellular dopamine and cAMP-regulated phosphoprotein of Mr 32 000 (DARPP-32) (DA- and cAMP-regulated phosphoprotein of Mr 32 kDa) at the threonine 75 residue, providing a compensatory signal to counteract DARPP-32/Thr34 (activated by protein kinase A), i.e. protein phosphatase-1 inhibition (Greengard 2001; Svenningsson et al. 2004). Also, Cdk5 appears to be essential in promoting the slow mode of compensatory endocytosis via rephosphorylation of endocytic proteins, such as dynamin (Tan et al. 2003; Tomizawa et al. 2003). However, pharmacological evidence suggests that Cdk5 plays the opposite role in neurons. Inhibition of Cdk5 enhanced neurotransmitter secretion in synaptosomal preparations (Tomizawa et al. 2002) and striatal slices (Chergui et al. 2004) suggests that while Cdk5 activation in non-neuronal cells leads to exocytosis, Cdk5 inhibition facilitates neurotransmitter release in neurons.
It is well documented that the loss of D3 DA receptors leads to excess synaptic DA and a hyperactive phenotype in mice, however the mechanism by which the D3 receptor affects DA release is unknown. Therefore, the first goal of this study was to establish an in vitro model in which we tested whether D3 DA receptor activation regulates K+-evoked [3H]DA release. The second goal was to investigate the subcellular mechanisms of D3 receptor-mediated autoregulation. To accomplish these goals, we created a PC-12 clone (PC-12/hD3) that expresses human D3 DA receptors with comparable binding affinity to the native receptor. This cell line was used as a model system to characterize regulatory mechanisms of D3 receptor-mediated [3H]DA release. The PC-12 cell line is a well-established model for studying neurosecretory mechanisms as it expresses various protein kinases and channels required for transmitter release (Ahnert-Hilger et al. 1987; Courtney et al. 1991). Our results demonstrate that D3 receptor activation inhibits K+-evoked [3H]DA release from PC-12/hD3 cells. This process appears to be governed by Cdk5 activation and upstream signaling that includes a Gi/oα protein, protein phosphatase 2B (PP-2B, calcineurin) and casein kinase 1 (CK1).
Materials and methods
Rat adrenal pheochromocytoma (PC-12) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cell culture reagents were obtained from Biological Industries (Kibbutz, Israel) or Sigma (St Louis, MO, USA). Bromocriptine, 3,6a,11, 14-tetrahydro-9-methoxy-2 methyl-(12H)-isoquino[1,2-b] pyrrolo[3,2-f][1,3] benzoxanzine-1-carboxylic acid (PD128907), sulpiride, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), raclopride, quinpirole, U99194 maleate, and olomoucine were purchased from Tocris (Avonmouth, UK). SB-277011-A was a gift from GlaxoSmithKline Pharmaceuticals (Harlow, Essex, UK). Roscovitine and 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) were purchased from Calbiochem (Darmstadt, Germany).
Cell culture and transfection
PC-12 cells were grown in a 5% CO2 humidified atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) and glutamine with 7.5% horse serum (Sigma), 7.5% fetal bovine serum (Biological Industries), 100 U/mL penicillin, and 100 mg/mL streptomycin (Biological Industries). Transient transfections were performed on a 70–80% confluent monolayer in 60-mm dishes, using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). PC-12 cells stably expressing the human D3 DA receptors (PC-12/hD3 cells) or vector control were generated by clonal selection after a Lipofectamine 2000-mediated transfection and were maintained in 800 μg/mL geneticin (G418) containing culture medium. The plasmid expressing the human D3 DA receptor was a gift from Dr Pierre Sokoloff (Department of Neurobiology and Molecular Pharmacology, Centre Paul Broca, Paris, France) and the coding region of the D3 receptor was subcloned into pcDNA3.
D3 DA receptor binding
Membrane fractions from PC-12/hD3 cells were prepared by homogenizing cells in a 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA, and 5 mM MgCl2. After centrifugation at 34 000 g for 30 min, pellets were resuspended in the original buffer and used for D3 receptor binding. The amount of membrane proteins in each assay was approximately 60–80 μg and the assay was incubated for 60 min at 25°C in a 96-well plate. Saturation binding of D3 receptors was performed in triplicate with 0.4–12.5 nM [3H]7-OH-DPAT (144 Ci/mmol; Amersham Biosciences, Piscataway, NJ, USA) in a final volume of 200 μL. Specific binding was defined as the difference between the binding of [3H]7-OH-DPAT in the absence and presence of 10 μM quinpirole. For competitive binding assays, displacement of [3H]7-OH-DPAT by test compounds (10 μM–0.1 nM) was carried out in the same assay buffer incubated with 2 nM [3H]7-OH-DPAT. To evaluate the effect of GTPγS on D3 receptor binding, a competition assay with various concentrations of bromocriptine (10 μM–0.1 nM) was incubated with 2 nM [3H]7-OH-DPAT in the presence or absence of 50 μM GTPγS. The incubation was terminated by filtering the membranes (Whatman GF/C filters) through a PHD™ cell harvester (Brandel, Gaithersberg, MD, USA). The filters were then washed three times with 5 mL of Tris–HCl buffer (pH 7.4) at 4°C and transferred to scintillation counting vials. Four milliliters of liquid scintillation cocktail (Beckman Ready Safe, Fullerton, CA, USA) was then added into each vial. The radioactivity in each sample was determined with a liquid scintillation analyzer (Packard, Meriden, CT, USA; 45–50% counting efficiency).
Measurement of cAMP
Stable PC-12/hD3 clones were cultured in 96-well microplates with cell titers of 104–106 cells/mL. Cells were incubated with 100 μL of D3 DA receptor agonists or antagonists in non-serum DMEM medium for 10 min. After aspirating excess medium, the cells were extracted (cAMP EIA assay kit; Biotrak, Amersham, Inc.) to determine the amount of intracellular cAMP as described in the manufacturer’s instructions (Protocol 3).
PC-12/hD3 cells were cultured in six-well plates until they reached 70–80% confluence at which point they were starved overnight in a serum-free culture medium. Cells were then treated with PD128907 for designated times from 0 to 60 min. Afterward, the medium was aspirated and the cells washed once with ice-cold phosphate-buffered saline (PBS) (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4, pH 7.4) and placed on ice. One percent sodium dodecyl sulfate buffer was directly added to culture wells, and the protein was extracted by sonicating to shear genomic DNA and boiling for 5 min. Small aliquots of the extracts were retained for protein determination by the bicinchoninic acid method (Pierce, Rockford, IL, USA) with bovine serum albumin as the standard. Equal amounts of protein (10–20 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10–12.5% polyacrylamide gels), and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were incubated overnight at 4°C with a primary antibody diluted in the Tris-buffered saline plus 0.1% Tween-20 (TBST). The antibody against pCdk5/Tyr15 (1 : 500 dilution) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against pAkt/Ser473 and pAkt/Thr308 (1 : 1000 dilution) were from Cell Signaling Technology (Beverly, MA, USA). The antibody against β-actin (1 : 1000 dilution) was from Sigma Co. The antibody against pDARPP-32/Thr75 (1 : 1000) was a kind gift from Drs Greengard and Nairn, and was also purchased from Cell Signaling Technology. After three 10-min washes in TBST buffer, blots were incubated for 1 h at 25°C with horseradish peroxidase-conjugated secondary antibodies in TBST buffer: 1 : 2000 goat anti-rabbit IgG (Amersham Pharmacia Biotech) for pAkt/Thr308 and pDARPP-32/Thr75; 1 : 2000 horse anti-mouse IgG (Amersham Pharmacia Biotech) for pAkt/Ser473; 1 : 4000 donkey anti-goat IgG (Amersham Pharmacia Biotech) for pCdk5. Finally, the blots were washed 3 × 10 min in TBST and developed using the enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech). The signals were exposed with Hyperfilm (Amersham Pharmacia Biotech) after which the blots were stripped and re-probed with anti-β-actin (1 : 1000; Sigma Co.) as a quantitative control. The resulting gel bands were quantified with a gel documentation system (Molecular Dynamics, Sunnyvale, CA, USA) and normalized to the corresponding controls.
Assessment of [3H]DA release from PC-12/hD3 cells
[3H]DA release from pre-loaded PC-12/hD3 cells was assayed in monolayer cultures at 37°C for the designated times (adapted from Tang et al. 1994). Briefly, cells were plated on poly-d-lysine (0.01 mg/mL)-coated six-well (800 000 cells/well) plates. At the time of the assay, the medium was removed by aspiration, and the cells were washed twice with 2 mL of Krebs-Ringer’s (KRS) buffer containing 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 26.2 mM NaHCO3, 10 mM glucose, and 1 mM ascorbic acid at pH 7.4. The basal or K+-evoked [3H]DA release was initiated by pre-loading [3H]DA (25 nM; final concentration) for 10 min, followed by three rapid washes (2 mL each) with KRS buffer at 25°C. Cells were then treated with KRS or 50 mM K+ in KRS buffer (K+-KRS) (NaCl was replaced with an equimolar concentration of KCl) in the presence or absence of the testing drugs. The incubation media were collected after 30 min incubation. In some experiments (Figs 1, 6a, 7 and 8a), to explore the pharmacological profile of drug-evoked [3H]DA release, every 5 min samples were collected for a total session of 30 min. Afterward, the media and cultured cells were solubilized in scintillation cocktail (Beckman Ready Safe), and radioactivity was determined by liquid scintillation spectrometry. The percentage of [3H]DA released in the medium was determined by dividing the radioactivity accumulated in the medium by total [3H]DA uptake in the cells (counts of released fractions plus the remaining from attached cells).
[3H]DA uptake assay
[3H]DA uptake by PC-12/hD3 cells was assessed in monolayer cultures at 70–80% confluency (similar to the [3H]DA release assay). The medium was removed by aspiration and cells were washed twice with Krebs-Ringer HEPES (KRH) buffer containing 130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.8 g/L glucose, and 10 mM HEPES (pH 7.4). Cells were then incubated in triplicate at 37°C in KRH buffer (2 mL/well) containing 100 μM pargyline, and 100 μM ascorbic acid in the absence or presence of 10 μM DA transporter blocker GBR12909 for 5 min. After another 10 min incubation with [3H]DA (25 nM) at 37°C, medium was aspirated and the cells were washed three times with ice-cold KRH buffer. The cells were then solubilized with 0.5 N NaOH and [3H]DA accumulation was quantified using a scintillation cocktail (Beckman Ready Safe). Specific DA uptake was determined by subtracting the amount of [3H]DA accumulated in the presence of 10 μM GBR12909.
In vitro kinase activity
After drug treatment, cells were extracted with lysis buffer (150 mM NaCl, 20 mM Tris–HCl, 1 mM EDTA, 0.5% Nonidet P-40, 5 mM dithiothreitol, 1 mM phenyl methylsulfonyl fluoride, 2.5 mM benzamidine, 20 μg/mL antipain, 20 μg/mL leupeptin, 5 μg/mL chymostatin, 5 μg/mL pepstatin, 50 mM NaF, and 5 mM Na3VO4 at pH 7.4). Cdk5 was immunoprecipitated using an anti-Cdk5 antibody conjugated to agarose beads (C8; Santa Cruz Biotechnology). The immunoprecipitated samples were resuspended and incubated with 2 μg of histone H1 (type II-S) (Sigma Co.) in 20 μL of kinase assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM dithiothreitol, 50 μM ATP, and 5 μCi [γ-32P] ATP) for 10 min at 30°C. The reaction was terminated by spotting 20 μL of the reaction mixture on a P81 phosphocellulose disc (Whatman P81; Upstate Biotechnology Inc., Lake Placid, NY, USA). The discs were washed three times (5 min each) with 0.75% phosphoric acid, followed by soaking in 95% ethanol for 5 min and dried in the air. The radioactivity was measured with a scintillation counter (Packard Inc.) and corrected with basal activity, measured at corresponding time but with boiled samples.
Confocal immunofluorescence imaging
PC12/hD3 cells were plated on sterile coverslips and starved with serum-free DMEM medium overnight. Cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS three times, permeabilized with cold methanol for 10 min and blocked with 5% non-fat milk for 30 min. The cells were then incubated with the pCdk5 antibody diluted in PBS followed by rhodamine conjugated anti-goat IgG (H + L) for red fluorescence (Jackson ImmunoResearch Laboratory, West Grove, PA, USA). The coverslips were washed twice with PBS and then mounted on glass slides. The images were monitored under a confocal microscope (Bio-rad Radiance 2100; Bio-rad, Hercules, CA, USA).
All the data were analyzed with the computer program GraphPad Prism™ (San Diego, CA, USA). The results are expressed as the mean ± SEM. The data were analyzed with a one-way anova followed by Dunnett′s post hoc multiple comparison test or a non-paired Student′s t-test. In some experiments (time-dependent [3H]DA release), a two-way anova was performed to detect significance between groups. The level of statistical significance was set at p <0.05.
Identification of DA receptors and DA transporter in PC-12 cells
We used a DA receptor binding assay to determine if there are endogenous DA D2-like receptors in parental PC-12 cells. [3H]raclopride binding indicated that there were trace amounts of D2-like receptors in PC-12 cells (7.14% specific binding defined by 10 μM sulpiride). To evaluate if DA transporters are expressed in the PC-12 cells, we determined the amount of [3H]DA uptake (radioactivity retained in the cell after 10 min incubation and washes; see ‘Materials and methods’ for details) in the absence and presence of a high-affinity DA uptake inhibitor, GBR12909. The results showed that the addition of 10 μM GBR12909 suppressed > 90% of DA uptake (control group: 100 ± 8.87% vs. GBR12909 treated group: 9.22 ± 1.67%, p <0.0001, unpaired t-test from three independent experiments) in PC-12 cells. Hence, the parent PC-12 cells had extremely low levels of endogenous D2-like receptors, but contained a substantial amount of functional DA transporters.
Functional characterization of human D3 DA receptor in PC-12/hD3 stable clone
Using G418 selection, we successfully cloned a PC-12 cell line stably expressing the human D3 DA receptor (pcDNA3/hD3 construct). The binding affinity was 1.61 nM, which is comparable to the binding affinity found in vivo using the D3 receptor-enriched rat limbic forebrain (Chiang et al. 2003). The Bmax value was 1171 fmol/mg protein, indicating a reasonable density for further experimentation. Furthermore, addition of D2/D3 receptor preferring agonists or antagonists (SB277011-A, bromocriptine, PD128907, sulpiride, 7-OH-DPAT, raclopride, quinpirole) dose-dependently inhibited the binding of [3H]7-OH-DPAT with various efficacies (data not shown), indicating that the D3 receptors in PC-12/hD3 cells displayed a normal pharmacophore. Cells treated with various D2/D3 agonists (5 μM of quinpirole, bromocriptine, DA, PD128907, or 7-OH-DPAT) inhibited forskolin-induced cAMP accumulation which could be prevented by the pre-treatment of D3 receptor preferring antagonists (SB27701-A or GR103691) further demonstrating functional receptor expression (Fig. S1).
D3 DA receptor-regulated K+-evoked [3H]DA release in PC-12/hD3 cells
Anatomical studies have shown that D3 DA receptors localize both pre-synaptically and post-synaptically (Diaz et al. 2000). The pre-synaptic receptors may function as autoreceptors to control release and/or synthesis of catecholamines (Richtand et al. 2001). To evaluate D3 receptors as autoreceptors controlling DA release, we pre-loaded PC-12/hD3 cells with [3H]DA followed with 50 mM KCl-KRS in the presence or absence of D3 preferring agonist PD128907. Addition of PD128907 dose-dependently (between 10 and 50 μM) inhibited K+-induced [3H]DA release, suggesting that D3 DA receptors act as autoreceptors to control neurotransmitter release (Fig. 1a). PD128907 did not have any measurable effect on basal [3H]DA release in parental PC-12 cells, control vector transfected PC-12, or PC12/hD3 cells (data not shown). Furthermore, inhibition of K+-evoked [3H]DA release by PD128907 was prevented by the pre-treatment of D3 preferring antagonists, SB27701-A or GR103691, indicating D3 receptor specificity (Fig. 1b).
Effect of pertussis toxin on D3 DA receptor-mediated autoinhibition of K+-evoked [3H]DA release
We next investigated if the observed inhibition is dependent on Gi/oα. We applied 100 ng/mL pertussis toxin (PTX) to cells overnight to ribosylate the α subunit of the Gi/o protein. Afterward we treated the cells with 50 μM PD128907 to evaluate the impact of Gi/oα on D3 receptor-mediated autoinhibition. PTX effectively blocked the inhibitory effect (Fig. 2). Therefore, we conclude that D3 receptor-mediated autoinhibition of DA release requires Gi/oα.
Signal transduction underlying the D3 receptor activation
Effects on Cdk5/DARPP-32 signaling were measured after treatment with 1 or 50 μM PD128907. At both dosages, D3 receptor activation led to Cdk5 and DARPP-32/Thr75 phosphorylation with peak activation approximately 10 min after the drug administration (Fig. 3; The 50 μM PD128907 dose is shown to match the [3H]DA release study). Confocal imaging confirmed that 10 min after D3 preferring agonist treatment, phosphoCdk5 signal intensified in cultured PC-12/hD3 cells (Fig. 4).
Stimulation of D3 receptors also triggered the phosphatidylinositol (PI)-3 kinase/Akt pathway. 50 μM PD128907 induced Akt phosphorylation at both Ser473 and Thr308 in PC-12/hD3 cells (Fig. 5). PhosphoAkt/Ser473 was increased approximately fourfold 20–25 min post-drug administration and was still elevated after 60 min. PhosphoAkt/Thr308 followed a similar pattern, with a maximal activation of about 2-fold 20–25 min post-drug administration.
Effect of Cdk5 and Akt signaling on D3 DA receptor-mediated autoinhibition of K+-evoked [3H]DA release
As we had found that D3 receptor activation leads to changes in the Cdk5/DARPP-32 and PI-3 kinase/Akt signaling pathways, we tested whether Cdk5 or PI-3 kinase blockade would affect the inhibitory effect of PD128907 on K+-evoked [3H]DA release. Addition of Cdk5 inhibitors roscovitine or olomoucine prevented DA release inhibition, as calculated by the 5 min level after various drug treatments (Fig. 6a). However, pre-treatment with the PI-3 kinase inhibitor wortmannin had no effect on PD128907-mediated inhibition of K+-evoked [3H]DA release (Fig. 6b). To confirm the role of Cdk5 in D3 receptor-mediated autoinhibition of [3H]DA release, PC-12/hD3 cells were transiently transfected with Cdk5 vector (full sequence construct; a gift from Dr Huang, Chung-Cheng University, Taiwan). Over-expression of Cdk5 in PC-12/hD3 cells potentiated the inhibitory effect of PD128907 on K+-evoked [3H]DA release (Fig. 7). Together, these data suggest that D3 receptor-dependent autoinhibition of DA release is dependent on the Cdk5 signaling pathway.
Effect of a CK1 inhibitor on D3 DA receptor-mediated autoinhibition of K+-evoked [3H]DA release
Because CK1 is known to regulate Cdk5 activity (Sharma et al. 1999; Liu et al. 2001), we evaluated the possible participation of CK1 in D3 receptor-mediated autoinhibition of [3H]DA release. Cells were pre-incubated with 1 or 10 μM of the CK1 inhibitor IC261 for 10 min followed by the addition of 50 μM PD128907 for another 10 min. Both doses of IC261 effectively prevented the inhibitory effect of PD128907 on K+-evoked [3H]DA release (Fig. 8a). In addition, treatment with IC261 or the D3 preferring antagonist SB277011-A significantly prevented the stimulatory effect of PD128907 on Cdk5 activity 5 min after drug treatment. The demonstrated mitigation of Cdk5 activity in this experiment provides further evidence of its importance in D3 autoreceptor signaling (Fig. 8b).
Effect of cyclosporin A on D3 DA receptor-mediated autoinhibition of K+-evoked [3H]DA release
Another regulator of Cdk5 activity is PP-2B, which dephosphorylates the C-terminal region of CK1 to increase its kinase activity (Liu et al. 2001). As illustrated in Fig. 9(a), pre-treatment of the PC-12/hD3 cells with cyclosporine A, an inhibitor of calcineurin, abolished D3 DA receptor-mediated DA release inhibition. Pre-treatment with cyclosporine A also blocked PD128907-induced phospho-Cdk5 increases in PC-12/hD3 cells (Fig. 9b), confirming the involvement of PP-2B in D3 DA receptor-mediated autoregulation.
In the current study, we demonstrate that in D3 DA receptor-expressing PC-12/hD3 cells, activation of D3 receptors by the D3 preferring agonist PD128907 significantly inhibits K+-evoked [3H]DA release. Activation of D3 receptors, in addition to the well-known inhibition of adenylyl cyclase, also induces intracellular Cdk5-DARPP-32/Thr75 and PI-3 kinase/Akt signaling. Interestingly, DA D3 receptor-mediated inhibition of K+-evoked DA release appears to be closely linked to the Cdk5 signaling pathway, as demonstrated by a series of experiments, including Cdk5 blockade with roscovitine or olomoucine, over-expression of Cdk5, and confocal imaging of phosphoCdk5. All these results suggest that D3 receptor-mediated Cdk5 activation/phosphorylation is responsible, at least in part, for the inhibition of K+-evoked [3H]DA release. Furthermore, D3 receptor-regulated DA release involves the participation of a Gi/oα subunit, CK1 and PP-2B. This study represents the first report that connects D3 DA receptors with down-stream signaling cascades involved in regulating DA release (a schematic diagram is illustrated in Fig. 10). Moreover, if the observed phenomena are applicable to other autoreceptors, the Cdk5-dependent signaling pathway might represent a unifying mediator for controlling pre-synaptic autoreceptor-regulated neurotransmitter release.
Stimulation of D3 DA receptors is known to initiate several intracellular signal pathways, resulting in inhibition of adenylyl cyclase V (Robinson and Caron 1997), activation of extracellular signal-regulated kinases 1 and 2 (Cussac et al. 1999), and triggering phospholipase C (PLC) and PKC-dependent cascades via Giα3 proteins (Pedrosa et al. 2004). In addition to intracellular signaling, activation of D3 receptors modulates ion channels on the cell membrane, i.e. activation of G-protein-coupled inward rectifier potassium channels and inhibition of P/Q-type calcium channels, possibly via a Gβγ protein (Kuzhikandathil and Oxford 1999). It has also been reported that the Cdk5/p35 complex phosphorylates P/Q-type voltage-dependent calcium channel. This action down-regulates channel activity and inhibits transmitter secretion (Tomizawa et al. 2002). In this study, we find that treatment with the D3 agonist PD128907 induces Cdk5 phosphorylation at Tyr15. This phosphorylation produces an active form of Cdk5, and concomitantly inhibits K+-evoked [3H]DA release. Further, we demonstrate that Cdk5 over-expression potentiates the PD128907-mediated inhibition of K+-evoked [3H]DA release in PC-12/hD3 cells. Conversely, inhibition of Cdk5 by either roscovitine or olomoucine effectively prevents the D3 receptor-mediated inhibition of [3H]DA release. Based on this evidence, we postulate that activation of D3 DA receptors leads to phosphorylation of P/Q type calcium channels via Cdk5 phosphorylation/activation, thereby decreasing the K+-evoked [3H]DA release. This molecular event, which underlies D3 receptor activation, appears to qualify D3 receptors as pre-synaptic autoreceptors, which function to modulate neurotransmitter release or neural activity (Joseph et al. 2002). Alternatively, an early study by Tang et al. (1994) indicated that activation of D3 receptors in heterogeneously expressed MN9D cells inhibited DA release via coupling to potassium channels. Whether D3 receptors activated Cdk5 signaling is responsible for P/Q-type calcium channel phosphorylation or potassium channel coupling requires further investigation.
Previous studies have reported that the catalytic subunit of Cdk5 is phosphorylated by CK1 and transformed into active Cdk5 (Sharma et al. 1999). We find that pre-treatment with the CK1 inhibitor IC261, which displays selectivity towards the CK1δ and CK1ε isoforms (Mashhoon et al. 2000), suppressed Cdk5 activity. At the same time, IC261 reversed the PD128907-mediated inhibition of K+-evoked [3H]DA release. It has been reported that CK1δ and CK1ε are regulated by autophosphorylation at the COOH termini. Elimination of autophosphorylation by either truncation of the COOH terminus or via dephosphorylation was found to increase CK1 activity (Liu et al. 2001). During a search for possible phosphatases associated with CK1 activation, a recent study reported that in neostriatal neurons, stimulation of metabotropic glutamate receptors activates CK1ε via the action of phosphatase PP-2B (Liu et al. 2001). In support of this, we find that pre-treatment with a PP-2B specific inhibitor, cyclosporine A, prevents the PD128907-inhibited K+-evoked [3H]DA release. This suggests that D3 receptors may somehow recruit PP-2B to dephosphorylate CK1, consequently affecting the Cdk5-dependent signal(s) that influence DA release.
Experiments relating to pre-treatment with PTX further implicate the α subunit of Gi/o as a potential transducer in D3 receptor-regulated [3H]DA release (Tang et al. 1994). Several previous studies have provided evidence associating up-stream regulators with synaptic transmitter release. First, stimulation of DA D2 receptors mobilizes intracellular Ca2+ stores via the PLC pathway, subsequently leading to a calcineurin (PP-2B)-dependent reduction in L-type currents (Hernandez-Lopez et al. 2000). Second, several studies demonstrate that a Gi/o-coupled receptor could also induce PLC activation, which hydrolyzes the membrane phospholipid PtdIns (4,5)P2 to increase the intracellular Ca2+ concentration and activate PKC activity (Schmidt et al. 1998; Murthy et al. 2004; Pedrosa et al. 2004). Hence, we speculate that activation of DA D3 receptor would also, indirectly, induce PLC to increase the intracellular Ca2+ concentration. Elevated intracellular Ca2+ would activate PP-2B activity and, consequently, dephosphorylate the COOH terminus of CK1. The activated CK1 would enhance the activity of downstream Cdk5 to phosphorylate and down-regulate the P/Q type calcium channel activity, resulting in a decrease in transmitter release.
Pre-synaptic DA autoreceptors are known to play an important role in the regulation of dopaminergic activity and have been implicated in behavioral aspects of drugs of abuse such as morphine, cocaine and methamphetamine (Richtand et al. 2001). In addition, dysregulation of brain Cdk5 and DARPP-32/Thr75 is believed to contribute to the development of drug addiction (Bibb et al. 2001; Chen and Chen 2005). Moreover, the PI-3 kinase/Akt pathway has been reported to be involved in mania as lithium, an effective anti-manic agent, effectively inhibits Akt activity (Beaulieu et al. 2004). The finding that the D3 receptor could be linked with Akt activity seems to strengthen the argument that a causal relationship between D3 DA receptors and mania exists. Overall, understanding the properties of D3 DA receptors and its signal transduction routes will provide new insights for developing therapeutic remedies against several DA-dependent brain disorders.
The authors thank Drs J. L. Maderdrut (Tulane University) and M. Calkins for editing the manuscript. The work was supported by Chang-Gung Memorial Hospital (CMRP1375III), the National Science Council (NSC93-2320-B-182-035) and the National Bureau of Controlled Drugs (DOH93-NNB-1026) in Taiwan.